The endosomal apparatus is a major site of membrane sorting in both endocytic and exocytic pathways. For ligand activated receptors it also provides a mechanism for achieving the proper balance of cell signaling from different membrane compartments. A main objective of this proposal is to understand how endocytosis controls cellular responses to the EGF receptor (EGFR), the prototype for the ErbB receptor family. The complexity of endocytic trafficking predicts that EGFRs use a multitude of distinct sorting signals at different locations in the cell. Characterization of these signals provides a framework for understanding regulation of EGFR transit in the endosomal apparatus. We have identified three non-overlapping sorting signals that control critical benchmarks in EGFR trafficking: 679-LL, which controls sorting in multivesicular endosome-to-lysosome transport intermediates or MVEs; 954-YLVI, which controls lysosomal sorting at a site intersecting the exocytic pathway; and 662-RxxxxPLTP, which controls sorting from endosomes to the plasma membrane. Completion of the proposed studies will provide new insights on the coordinated action of these signals during ligand-regulated EGFR trafficking. Since related ErbB receptors have divergent sorting signals compared to EGFR, we will also gain insight to how ErbB signaling in general is controlled by intracellular trafficking. This is particularly important for ErbB2, whose ligand-dependent activation by EGFR requires EGFR sequences that regulate post-endocytic trafficking and not intrinsic tyrosine kinase activity. The availability of dominant-inhibitory mutations that disrupt EGFR trafficking at defined steps also provides a unique set of reagents with which to study the temporal and spatial organization of signaling from endosomes. The following hypotheses will be tested: 1. Ligand-induced EGFR post-endocytic sorting to lysosomes is mediated by the concerted action of multiple sorting signals acting in a step-wise fashion. 2. The 679-LL MVE sorting signal is recognized as part of a larger motif whose physiological function is dependent on protein-protein interactions. 3. The 662-RxxxxPLTP sorting signal controls EGFR recycling to the plasma membrane, and is regulated by MAP kinase-mediated phosphorylation of Thr669. 4. The 679-LL sorting signal regulates EGFR signaling by controlling the balance of growth stimulatory pathways elicited during endocytosis. This hypothesis is based on new data suggesting that EGFRs with an inactivating L679A,L680A mutation selectively activate survival pathways compared to wild-type, and will be tested using both in vitro and in vivo experimental models.